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Aberration‐corrected scanning transmission electron microscopy (STEM) has been advancing resolution, sensitivity, and microanalysis due to the intense demands of atomic‐level microstructural investigations. Recent STEM technologies require preparing a thin lamella whose thickness is ideally below 20 nm. Although focused‐ion‐beam/scanning‐electron‐microscopy (FIB/SEM) is an established method to prepare a high‐quality lamella, nanometer‐level controllability of lamella thickness remains a fundamental problem. Here, the robust preparation of a sub‐20‐nm‐thin lamella is demonstrated by FIB/SEM with real‐time feedback from thickness quantification. The lamella thickness is quantified by back‐scattered‐electron SEM imaging in a thickness range between 0 and 100 nm without any reference to numerical simulation. Using real‐time feedback from the thickness quantification, the FIB/SEM terminates thinning a lamella at a targeted thickness. The real‐time feedback system eventually provides 1‐nm‐level controllability of the lamella thickness. As a proof‐of‐concept, a near‐10‐nm‐thin lamella is prepared from a SrTiO3 crystal by our methodology. Moreover, the lamella thickness is controllable at a target heterointerface. Thus, a sub‐20‐nm‐thin lamella is prepared from a LaAlO3/SrTiO3 heterointerface. The methodology offers a robust and operator‐independent platform to prepare a sub‐20‐nm‐thin lamella from various materials. This platform will broadly impact aberration‐corrected STEM studies in materials science and the semiconductor industry. [ABSTRACT FROM AUTHOR]

Male infertility is a significant factor in approximately half of all infertility cases and is marked by a decreased sperm count and motility. A decreased sperm count is caused by not only a decreased production of sperm but also decreased numbers successfully passing through the male reproductive tract. Smooth muscle movement may play an important role in sperm transport in the male reproductive tract; thus, understanding the mechanism of this movement is necessary to elucidate the cause of sperm transport disorder. Recent studies have highlighted the presence of platelet-derived growth factor receptor α (PDGFRα)-positive interstitial cells (PICs) in various smooth muscle organs. Although research is ongoing, PICs in the male reproductive tract may be involved in the regulation of smooth muscle movement, as they are in other smooth muscle organs. This review summarizes the findings to date on PICs in male reproductive organs. Further exploration of the structural, functional, and molecular characteristics of PICs could provide valuable insights into the pathogenesis of male infertility and potentially lead to new therapeutic approaches. [ABSTRACT FROM AUTHOR]

Focused ion beam (FIB) has become a powerful tool for transmission electron microscopy sample preparation in the nanoelectronics industry and has in recent years also shown its benefits for specific preparation steps in electrical scanning probe microscopy (SPM). Most recently, a novel SPM approach – so-called reverse tip sample (RTS) SPM – has been proposed in which the position of sample and tip are switched compared to standard SPM; in RTS SPM the sample is attached to the end of a cantilever beam. To achieve this configuration, the region of interest must first be extracted from a substrate and then needs to be reliably fixed to the cantilever by FIB. Therefore, we have explored and developed dedicated FIB preparation methods for RTS SPM in this work. Our established procedures ensure a strong mechanical and good electrical connection of the sample to the cantilever for both cross-section and top view sample preparation. Furthermore, we introduce an approach for mounting samples from a full wafer size workflow. This paper presents the developed FIB procedures and discusses the quality and stability of all mounted samples and their electrical evaluation in RTS SPM.

In this work, a novel crystal growth method termed suspended drop crystallization has been developed. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an electron microscopy grid without any additional support layers. The grid is then suspended within a crystallization chamber designed in-house, allowing for vapor diffusion to occur from both sides of the drop. A UV-transparent window above and below the grid enables the monitoring of crystal growth via light, UV or fluorescence microscopy. Once crystals have formed, the grid can be removed and utilized for X-ray crystallography or microcrystal electron diffraction (MicroED) directly without having to manipulate the crystals. To demonstrate the efficacy of this method, crystals of the enzyme proteinase K were grown and its structure was determined by MicroED following focused ion beam/scanning electron microscopy milling to render the sample thin enough for cryoEM. Suspended drop crystallization overcomes many of the challenges associated with sample preparation, providing an alternative workflow for crystals embedded in viscous media, sensitive to mechanical stress and/or subject to preferred orientation on electron microscopy grids.

A structure of the murine voltage-dependent anion channel (VDAC) was determined by microcrystal electron diffraction (MicroED). Microcrystals of an essential mutant of VDAC grew in a viscous bicelle suspension, making it unsuitable for conventional X-ray crystallography. Thin, plate-like crystals were identified using scanning-electron microscopy (SEM). Crystals were milled into thin lamellae using a focused-ion beam (FIB). MicroED data were collected from three crystal lamellae and merged for completeness. The refined structure revealed unmodeled densities between protein monomers, indicative of lipids that likely mediate contacts between the proteins in the crystal. This body of work demonstrates the effectiveness of milling membrane protein microcrystals grown in viscous media using a focused ion beam for subsequent structure determination by MicroED. This approach is well suited for samples that are intractable by X-ray crystallography. To our knowledge, the presented structure is a previously undescribed mutant of the membrane protein VDAC, crystallized in a lipid bicelle matrix and solved by MicroED.

Male infertility is a significant factor in approximately half of all infertility cases and is marked by a decreased sperm count and motility. A decreased sperm count is caused by not only a decreased production of sperm but also decreased numbers successfully passing through the male reproductive tract. Smooth muscle movement may play an important role in sperm transport in the male reproductive tract; thus, understanding the mechanism of this movement is necessary to elucidate the cause of sperm transport disorder. Recent studies have highlighted the presence of platelet-derived growth factor receptor α (PDGFRα)-positive interstitial cells (PICs) in various smooth muscle organs. Although research is ongoing, PICs in the male reproductive tract may be involved in the regulation of smooth muscle movement, as they are in other smooth muscle organs. This review summarizes the findings to date on PICs in male reproductive organs. Further exploration of the structural, functional, and molecular characteristics of PICs could provide valuable insights into the pathogenesis of male infertility and potentially lead to new therapeutic approaches.

ABSTRACT The marine, bloom-forming dinoflagellate Prorocentrum cordatum CCMP 1329 (formerly P. minimum) has a genome atypical of eukaryotes, with a large size of ~4.15 Gbp, organized in plentiful, highly condensed chromosomes and packed in a dinoflagellate-specific nucleus (dinokaryon). Here, we apply microscopic and proteogenomic approaches to obtain new insights into this enigmatic nucleus of axenic P. cordatum. High-resolution focused ion beam/scanning electron microscopy analysis of the flattened nucleus revealed highest density of nuclear pores in the vicinity of the nucleolus, a total of 62 tightly packed chromosomes (~0.4–6.7 µm3), and interaction of several chromosomes with the nucleolus and other nuclear structures. A specific procedure for enriching intact nuclei was developed to enable proteomic analyses of soluble and membrane protein-enriched fractions. These were analyzed with geLC and shotgun approaches employing ion-trap and timsTOF (trapped-ion-mobility-spectrometry time-of-flight) mass spectrometers, respectively. This allowed identification of 4,052 proteins (39% of unknown function), out of which 418 were predicted to serve specific nuclear functions; additional 531 proteins of unknown function could be allocated to the nucleus. Compaction of DNA despite very low histone abundance could be accomplished by highly abundant major basic nuclear proteins (HCc2-like). Several nuclear processes including DNA replication/repair and RNA processing/splicing can be fairly well explained on the proteogenomic level. By contrast, transcription and composition of the nuclear pore complex remain largely elusive. One may speculate that the large group of potential nuclear proteins with currently unknown functions may serve yet to be explored functions in nuclear processes differing from those of typical eukaryotic cells. IMPORTANCE Dinoflagellates form a highly diverse group of unicellular microalgae. They provide keystone species for the marine ecosystem and stand out among others by their very large, unusually organized genomes embedded in the nuclei markedly different from other eukaryotic cells. Functional insights into nuclear and other cell biological structures and processes of dinoflagellates have long been hampered by the paucity of available genomic sequences. The here studied cosmopolitan P. cordatum belongs to the harmful algal bloom-forming, marine dinoflagellates and has a recently de novo assembled genome. We present a detailed 3D reconstruction of the P. cordatum nucleus together with comprehensive proteogenomic insights into the protein equipment mastering the broad spectrum of nuclear processes. This study significantly advances our understanding of mechanisms and evolution of the conspicuous dinoflagellate cell biology.

Morphological analysis of organelles is one of the important clues for understanding the cellular conditions and mechanisms occurring in cells. In particular, nanoscale information within crowded intracellular organelles of tissues provide more direct implications when compared to analyses of cells in culture or isolation. However, there are some difficulties in detecting individual shape using light microscopy, including super-resolution microscopy. Transmission electron microscopy (TEM), wherein the ultrastructure can be imaged at the membrane level, cannot determine the whole structure, and analyze it quantitatively. Volume EM, such as focused ion beam/scanning electron microscopy (FIB/SEM), can be a powerful tool to explore the details of three-dimensional ultrastructures even within a certain volume, and to measure several parameters from them. In this review, the advantages of FIB/SEM analysis in organelle studies are highlighted along with the introduction of mitochondrial analysis in injured motor neurons. This would aid in understanding the morphological details of mitochondria, especially those distributed in the cell bodies as well as in the axon initial segment (AIS) in mouse tissues. These regions have not been explored thus far due to the difficulties encountered in accessing their images by conditional microscopies. Some mechanisms of nerve regeneration have also been discussed with reference to the obtained findings. Finally, future perspectives on FIB/SEM are introduced. The combination of biochemical and genetic understanding of organelle structures and a nanoscale understanding of their three-dimensional distribution and morphology will help to match achievements in genomics and structural biology. [ABSTRACT FROM AUTHOR]

The electrochemical reduction of CO2 provides a pathway to a sustainable carbon cycle, allowing for the production of hydrocarbons critical for both chemical industry and mobility. Major engineering challenges need to be met in order to achieve efficient and large‐scale CO2 electrolysis. One central challenge is to find the optimum structure for the gas diffusion electrode at the heart of the electrolyzer. An optimum structure will achieve higher conversion efficiencies, lifetime, and product selectivity at lower cost. Critical structural properties of the electrode span the scale from nanometers to millimeters. To rationalize the optimization process, it behooves us to obtain a fully resolved multi‐scale model of the electrode. This digital twin, produced by bridging scale and employing multiple imaging methods, enables the digital study, simulation, and modification of the structure of the electrode. The model is used to simulate the transport processes vital to the functioning of the electrode thus further advancing the digital twin. Subsequently, it is explored how changes to the structure affect the predicted transport properties. The digital twin presented is only supposed to be the kernel and will be complemented by numerous future works. [ABSTRACT FROM AUTHOR]

Functional and structural studies investigating macroscopic connectivity in the human cerebral cortex suggest that high-order associative regions exhibit greater connectivity compared to primary ones. However, the synaptic organization of these brain regions remains unexplored. In the present work, we conducted volume electron microscopy to investigate the synaptic organization of the human brain obtained at autopsy. Specifically, we examined layer III of Brodmann areas 17, 3b, and 4, as representative areas of primary visual, somatosensorial, and motor cortex. Additionally, we conducted comparative analyses with our previous datasets of layer III from temporopolar and anterior cingulate associative cortical regions (Brodmann areas 24, 38, and 21). 9,690 synaptic junctions were 3D reconstructed, showing that certain synaptic characteristics are specific to particular regions. The number of synapses per volume, the proportion of the postsynaptic targets, and the synaptic size may distinguish one region from another, regardless of whether they are associative or primary cortex. By contrast, other synaptic characteristics were common to all analyzed regions, such as the proportion of excitatory and inhibitory synapses, their shapes, their spatial distribution, and a higher proportion of synapses located on dendritic spines. The present results provide further insights into the synaptic organization of the human cerebral cortex., (© The Author(s) 2024. Published by Oxford University Press.)

To understand general features in evolution of kinetochore organization, investigating a wide range of mitotic mechanisms in various non-model eukaryotes is necessary. A binucleate flagellate Giardia intestinalis is a representative of highly divergent eukaryotic lineage of Metamonads. FIB/SEM tomography was used to investigate ultrastructural details of its mitotic architecture, including kinetochores. Giardia undergoes semi-open mitosis, with the nuclear envelope remaining intact except for polar fenestrae, allowing microtubules to enter the nucleoplasm. At the onset of mitosis, the nuclear envelope bends inward, forming a concave depression at the spindle poles. Spindle microtubules emanate from a cytoplasmic fuzzy microtubule organizing center near the flagellar basal bodies. Kinetochoral microtubules enter the nucleoplasm and bind to kinetochores. A small bipartite kinetochore composed of a dense inner disk, approximately 46 nm in diameter, and a two-armed outer fork, is attached to just one microtubule. To our knowledge, this is the first in situ evidence of a one-microtubule attachment to a kinetochore, which could represent a basic eukaryotic situation.

Small-conductance calcium-activated potassium (SK) channels are crucial for learning and memory. However, many aspects of their spatial organization in neurons are still unknown. In this study, we have taken a novel approach to answering these questions combining a pre-embedding immunogold labeling with an automated dual-beam electron microscope that integrates focused ion beam milling and scanning electron microscopy (FIB/SEM) to gather 3D map ultrastructural and biomolecular information simultaneously. Using this new approach, we evaluated the number and variability in the density of extrasynaptic SK2 channels in 3D reconstructions from six dendritic segments of excitatory neurons and six inhibitory neurons present in the stratum radiatum of the CA1 region of the mouse. SK2 immunoparticles were observed throughout the surface of hippocampal neurons, either scattered or clustered, as well as at intracellular sites. Quantitative volumetric evaluations revealed that the extrasynaptic SK2 channel density in spines was seven times higher than in dendritic shafts and thirty-five times higher than in interneurons. Spines showed a heterogeneous population of SK2 expression, some spines having a high SK2 content, others having a low content and others lacking SK2 channels. SK2 immunonegative spines were significantly smaller than those immunopositive. These results show that SK2 channel density differs between excitatory and inhibitory neurons and demonstrates a large variability in the density of SK2 channels in spines. Furthermore, we demonstrated that SK2 expression was associated with excitatory synapses, but not with inhibitory synapses in CA1 pyramidal cells. Consequently, regulation of excitability and synaptic plasticity by SK2 channels is expected to be neuron class- and target-specific. These data show that immunogold FIB/SEM represent a new powerful EM tool to correlate structure and function of ion channels with nanoscale resolution. [ABSTRACT FROM AUTHOR]

The axon initial segment (AIS) is structurally and functionally distinct from other regions of the axon, yet alterations in the milieu of the AIS after brain injury have not been well characterized. In this study, we have examined extracellular and intracellular changes in the AIS after hypoglossal nerve injury. Microglial adhesions to the AIS were rarely observed in healthy controls, whereas microglial adhesions to the AIS became apparent in the axonal injury model. Regarding intra‐AIS morphology, we focused on mitochondria because mitochondrial flow into the injured axon appears critical for axonal regeneration. To visualize mitochondria specifically in injured axons, we used Atf3:BAC transgenic mice whose mitochondria were labeled with GFP in response to nerve injury. These mice clearly showed mitochondrial localization in the AIS after nerve injury. To precisely confirm the light microscopic observations, we performed three‐dimensional ultrastructural analysis using focused ion beam/scanning electron microscopy (FIB/SEM). Although the healthy AIS was not surrounded by microglia, tight microglial adhesions with thick processes adhering to the AIS were observed after injury. FIB/SEM simultaneously allowed the observation of mitochondrial localization in the AIS. In the AIS of non‐injured neurons, few mitochondria were observed, whereas mitochondria were abundantly localized in the cell body, axon hillock, and axon. Intriguingly, in the injured AIS, numerous mitochondria were observed throughout the AIS. Taken together, axonal injury changes the extracellular glial environment surrounding the AIS and intracellular mitochondrial localization in the AIS. These changes would be crucial responses, perhaps for injured neurons to regenerate after axonal injury. [ABSTRACT FROM AUTHOR]

Cryo-electron tomography (cryo-ET) has the potential to revolutionize our understanding of the building blocks of life since it provides the unique opportunity to study molecules and membrane architectures in the context of cellular interaction. In particular, the combination of fluorescence imaging with focused ion beam (FIB) milling allows the targeting of specific structures in thick cellular samples by preparing thin lamellae that contain a specific fluorescence marker. This technique has conventionally been time-consuming, as it requires sample transfer to multiple microscopes and presents several technical challenges that currently limit its success. Here we describe METEOR, a FIB-integrated microscopy solution that streamlines the correlative cryo-ET workflow. It protects the sample from ice contamination by minimizing handling steps, thus increasing the likelihood of high-quality data. It also allows for monitoring of the milling procedure to ensure the molecule of interest is captured and can then be imaged by cryo-ET. [ABSTRACT FROM AUTHOR]

[Display omitted] The palette of mineralized tissues in fish is wide, and this is particularly apparent in fish dentin. While the teeth of all vertebrates except fish contain a single dentinal tissue type, called orthodentin, dentin in the teeth of fish can be one of several different tissue types. The most common dentin type in fish is orthodentin. Orthodentin is characterized by several key structural features that are fundamentally different from those of bone and from those of osteodentin. Osteodentin, the second-most common dentin type in fish (based on the tiny fraction of fish species out of ∼30,000 extant fish species in which tooth structure was so far studied), is found in most Selachians (sharks and rays) as well as in several teleost species, and is structurally different from orthodentin. Here we examine the hypothesis that osteodentin is similar to anosteocytic bone tissue in terms of its micro- and nano-structure. We use Focused Ion Beam-Scanning Electron Microscopy (FIB/SEM), as well as several other high-resolution imaging techniques, to characterize the 3D architecture of the three main components of osteodentin (denteons, inter-denteonal matrix, and the transition zone between them). We show that the matrix of osteodentin, although acellular, is extremely similar to mammalian osteonal bone matrix, both in general morphology and in the three-dimensional nano-arrangement of its mineralized collagen fibrils. We also document the presence of a complex network of nano-channels, similar to such networks recently described in bone. Finally, we document the presence of strings of hyper-mineralized small 'pearls' which surround the denteonal canals, and characterize their structure. [ABSTRACT FROM AUTHOR]

Since the second half of the XX century, primer mixtures based on mercury fulminate have become a rare occurrence on small ammunition markets in Western Europe and North America. As a consequence, Hg-containing gunshot residue (GSR) particles have not been as deeply investigated as residues from lead-based primer mixtures. As a matter of fact, no mention of GSR particles from mercuric primers is made by the current ASTM standard procedure for gunshot residue analysis. However, those laboratories dealing with ammunition and firearms produced in Eastern Europe or Asia still have a forensic interest in Hg-containing GSR. In this paper, a brief description of chemical composition and inner morphology of GSR particles from three different mercuric primers is reported. Regarding composition, arguments are given to promote SbSnHg residues to Characteristic of GSR particles when mercuric primers are discharged. From a morphological point of view, presence of inner nodules and other inhomogeneities were shown in GSR particles milled in a FIB/SEM. Moreover, mercury vaporization under the electron beam was observed for a particle reduced to a lamella. Mercury evanescence in GSR was interpreted in terms of mercury segregation during particle formation and higher mobility of Hg atoms in presence of defects (vacancies) in a strained lattice. [Display omitted] • Mercuric primers give origin to Hg-containing GSR particles. • SbSnHg can be considered Characteristic of GSR particles from mercuric primers. • Hg-containing GSR particles are neither homogeneous nor necessarily amorphous. • Hg vaporization under the electron beam was documented for a GSR milled into a lamella. • Mercury evanescence was interpreted in terms of Hg segregation during particle formation. [ABSTRACT FROM AUTHOR]

Abstract Background Particokinetic models are important to predict the effective cellular dose, which is key to understanding the interactions of particles with biological systems. For the reliable establishment of dose–response curves in, e.g., the field of pharmacology and toxicology, mostly the In vitro Sedimentation, Diffusion and Dosimetry (ISDD) and Distorted Grid (DG) models have been employed. Here, we used high resolution scanning electron microscopy to quantify deposited numbers of particles on cellular and intercellular surfaces and compare experimental findings with results predicted by the ISDD and DG models. Results Exposure of human lung epithelial A549 cells to various concentrations of differently sized silica particles (100, 200 and 500 nm) revealed a remarkably higher dose deposited on intercellular regions compared to cellular surfaces. The ISDD and DG models correctly predicted the areal densities of particles in the intercellular space when a high adsorption (“stickiness”) to the surface was emulated. In contrast, the lower dose on cells was accurately inferred by the DG model in the case of “non-sticky” boundary conditions. Finally, the presence of cells seemed to enhance particle deposition, as aerial densities on cell-free substrates were clearly reduced. Conclusions Our results further validate the use of particokinetic models but also demonstrate their limitations, specifically, with respect to the spatial distribution of particles on heterogeneous surfaces. Consideration of surface properties with respect to adhesion and desorption should advance modelling approaches to ultimately predict the cellular dose with higher precision.

Alzheimer's disease is the most common form of dementia, characterized by a persistent and progressive impairment of cognitive functions. Alzheimer's disease is typically associated with extracellular deposits of amyloid-β peptide and accumulation of abnormally phosphorylated tau protein inside neurons (amyloid-β and neurofibrillary pathologies). It has been proposed that these pathologies cause neuronal degeneration and synaptic alterations, which are thought to constitute the major neurobiological basis of cognitive dysfunction in Alzheimer's disease. The hippocampal formation is especially vulnerable in the early stages of Alzheimer's disease. However, the vast majority of electron microscopy studies have been performed in animal models. In the present study, we performed an extensive 3D study of the neuropil to investigate the synaptic organization in the stratum pyramidale and radiatum in the CA1 field of Alzheimer's disease cases with different stages of the disease, using focused ion beam/scanning electron microscopy (FIB/SEM). In cases with early stages of Alzheimer's disease, the synapse morphology looks normal and we observed no significant differences between control and Alzheimer's disease cases regarding the synaptic density, the ratio of excitatory and inhibitory synapses, or the spatial distribution of synapses. However, differences in the distribution of postsynaptic targets and synaptic shapes were found. Furthermore, a lower proportion of larger excitatory synapses in both strata were found in Alzheimer's disease cases. Individuals in late stages of the disease suffered the most severe synaptic alterations, including a decrease in synaptic density and morphological alterations of the remaining synapses. Since Alzheimer's disease cases show cortical atrophy, our data indicate a reduction in the total number (but not the density) of synapses at early stages of the disease, with this reduction being much more accentuated in subjects with late stages of Alzheimer's disease. The observed synaptic alterations may represent a structural basis for the progressive learning and memory dysfunctions seen in Alzheimer's disease cases. [ABSTRACT FROM AUTHOR]

A novel method for the preparation of lamellas made from porous and brittle compressed green powder using a focused ion beam (FIB) is described. One of the main purposes for the development of this methodology is to use this type of samples in micro-electromechanical systems (MEMS) chips for in situ transmission electron microscopy heating/biasing experiments, concomitant with maintaining the mechanical integrity and the absence of contamination of samples. This is accomplished through a modification of the standard FIB procedure for the preparation of lamellas, the adaptation of conventional chips, as well as the specific transfer of the lamella onto the chips. This method is versatile enough to be implemented in most commercially available FIB systems and MEMS chips., Competing Interests: Conflict of Interest The authors declare that they have no competing interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Microscopy Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Purpose: The inverted internal limiting membrane (ILM) technique was recently introduced for refractory macular hole. Here, we evaluate a case of macular pucker formation after macular hole surgery using the inverted ILM flap technique and silicone oil tamponade. After undergoing vitrectomy combined with ILM removal, the patient had a good visual prognosis. Observations: A 49-year-old male with macular hole affecting both of his eyes underwent vitrectomies. Three months after the first surgery in his right eye, macular pucker formation was observed in the macula, which was associated with the ILM flap used to cover the macular hole. After peeling the ILM, the macula returned to a normal contour and visual acuity improved. Examination of the removed ILM revealed macrophage-like cells containing silicone oil particles that were responsible for the ILM contraction. Conclusions and Importance: When using the inverted ILM flap technique and silicone oil, macular pucker may occur after macular hole surgery. Peeling of the ILM flap restored the macular shape and did not reopen the macular hole, thereby improving visual acuity. Thus, silicone oil should be used with caution when performing macular hole surgery with the ILM flap technique.

Members of the family Meloidae are known to produce cantharidin, a highly toxic monoterpene found in their hemolymph and exuded as droplets capable of deterring many predators. As a nuptial gift, males transfer large amounts of cantharidin to females via a spermatophore, which is formed by specific accessory glands containing high concentrations of this terpene. Using light, electron and ion beam microscopy, the ultrastructural features of the three pairs of male accessory glands as well as the glandular part of the vasa deferentia were comparatively investigated in seven species of blister beetles belonging to five different tribes and two subfamilies. All gland pairs examined share common features such as mesodermal derivation, the presence of muscle sheath, a developed rough endoplasmic reticulum, abundant mitochondria, secretory vesicles, and microvillated apical membranes. Within the same species, glands exhibit distinctive features, suggesting that each pair is responsible for the formation of a specific substance. The vasa deferentia, while showing many similarities within the family, often exhibit features unique to each of the individual species investigated, whereas the accessory glands of the first and second pairs display the highest degree of ultrastructural variability. A comparison across the species shows an interesting constancy limited to ultrastructural features in the third pair of accessory glands. The similarities and differences among the species are discussed in the light of the available literature and in relation to the potential role that blister beetles’ male accessory glands could play in the storage and management of cantharidin.

Purpose: The formation of macular hole after receiving anti-vascular endothelial growth factor (anti-VEGF) therapy is rare. We report a case of macular hole that occurred after intravitreal injection of an anti-VEGF agent for age-related macular degeneration (AMD) in a patient, who underwent vitrectomy combined with choroidal neovascularization (CNV) removal. Observations: A 64-year-old female with AMD affecting her right eye received an intravitreal injection of an anti-VEGF agent. After treatment, we identified a full thickness macular hole (MH) that was associated with the rapid resolution of the macular edema and contraction of the CNV. After performing vitrectomy combined with CNV removal, the MH closed and her visual acuity improved. Examination of the removed CNV revealed a network of microvessels devoid of pericytes. Conclusions and importance: The present findings suggest that rapid resolution of macular edema and contraction of the CNV and/or mild increase in the vitreous traction after anti-VEGF therapy could potentially cause MH. CNV removal via the MH may be an acceptable procedure, if the MH remains open, the CNV is of the classic type, and it spares a central portion of the fovea.

Abstract Synaptic dysfunction or loss in early stages of Alzheimer’s disease (AD) is thought to be a major structural correlate of cognitive dysfunction. Early loss of episodic memory, which occurs at the early stage of AD, is closely associated with the progressive degeneration of medial temporal lobe (MTL) structures of which the transentorhinal cortex (TEC) is the first affected area. However, no ultrastructural studies have been performed in this region in human brain samples from AD patients. In the present study, we have performed a detailed three-dimensional (3D) ultrastructural analysis using focused ion beam/scanning electron microscopy (FIB/SEM) to investigate possible synaptic alterations in the TEC of patients with AD. Surprisingly, the analysis of the density, morphological features and spatial distribution of synapses in the neuropil showed no significant differences between AD and control samples. However, light microscopy studies showed that cortical thickness of the TEC was severely reduced in AD samples, but there were no changes in the volume occupied by neuronal and glial cell bodies, blood vessels, and neuropil. Thus, the present results indicate that there is a dramatic loss of absolute number of synapses, while the morphology of synaptic junctions and synaptic spatial distribution are maintained. How these changes affect cognitive impairment in AD remains to be elucidated.

Conventional imaging for three-dimensional (3D) ultra-architectural analysis of collagen fibers and fibroblasts is time-consuming and requires numerous ultrathin sections to search the target area. Currently, no method allows 3D ultra-architectural analysis of predetermined areas including spatial relationships between collagen fibers and fibroblasts in vitro. Herein, we developed a new method for in vitro analysis of the 3D ultrastructure of fibroblasts and collagen fibers using CLEM optimized for picrosirius red staining and FIB/SEM tomography. Collagen fibers were observed between, rather than on top of, stacked cells. This method offers the advantage of mesoscopic and ultrastructural analysis, thus minimizing bias and ensuring accurate observation. [ABSTRACT FROM AUTHOR]

Neurons in the central nervous system display a great diversity of synaptic architecture. While much of our knowledge on the excitatory synapse morphology derives from the prototypical asymmetric synapses, little has been studied about the atypical crest-type synapse that exists in the restricted brain regions. Here, we used focused ion beam scanning electron microscopy (FIB/SEM) to image a neuropil volume of interpeduncular nucleus (IPN) and manually reconstructed several dendrites to obtain an insight about the topography and quantitative features of crest synapses. Three-dimensional reconstruction showed numerous U-shaped structures protruding from the IPN dendrites. On either faces of the U-shaped structure, a pair of crest synapses are aligned in parallel such that there exists a positive correlation between the postsynaptic density (PSD) area of synapses that participate in pair formation. Interestingly, mitochondria are excluded from the site of crest synapses. Several presynaptic axons run through the hollow, cylindrical space of the U-shape grooves such that the plasma membrane of the axon and the dendrite are organized in a tight opposition without any intervening glial membrane. Unlike the peculiar dendritic morphology, IPN neurons possess typical somatic morphology with an oval, centrally located nucleus. In conclusion, our data reveals a hitherto unknown unique topographical feature of crest synapses in the IPN. • IPN dendrites possess numerous U-shaped structures. • Crest-type synapses are organized in pairs. • Postsynaptic density areas of crest synapses in pairs are positively correlated. • Mitochondria are excluded from the site of crest synapses. • Axons run through the hollow space in IPN dendrites. [ABSTRACT FROM AUTHOR]

Cementocytes in cementum form a lacuna-canalicular network. However, the 3D ultrastructure and range of the cementocyte network are unclear. Here, the 3D ultrastructure of the cementocyte network at the interface between cementum and periodontal ligament (PDL) was investigated on the mesoscale using FIB/SEM tomography. The results revealed a cellular network of cementocytes and PDL cells. A previous histomorphological study revealed the osteocyte-osteoblast-PDL cellular network. We extended this knowledge and revealed the cementum-PDL-bone cellular network, which may orchestrate the remodeling and modification of periodontal tissue, using a suitable method for imaging of complex tissue. [ABSTRACT FROM AUTHOR]

The periodontal ligament (PDL) is a unique connective tissue mainly comprising collagen fiber bundles and cells between the roots of teeth and inner walls of the alveolar-bone socket. PDL fiber bundles are arrayed between teeth and bone, with both ends embedded in the cementum or alveolar bone as Sharpey's fiber. These bundles, synthesized by PDL fibroblasts (PDLFs), form several distinct groups within the PDL which has important functions besides tooth anchoring including tooth nutrition, proprioception, sensory detection, homoeostasis, and repair of damaged tissue. However, little is known about how the regular-PDL fiber bundle arrays are formed, maintained, and remodeled over large distances from cementum to alveolar bone. Recently, novel instruments and 3D-imaging methods have been developed that have been applied to the investigation of hard tissues including the PDL. Work from our laboratory has revealed the three-dimensional (3D) ultrastructure of PDLFs and PDL collagen bundles by focused ion beam/scanning electron microscope tomography. We have shown that PDLFs have a flat shape with long processes or a wing-like shape, while PDL bundles are a multiple-branched structure wrapped in thin sheets of PDLF cytoplasm. Furthermore, PDLFs form an extensive cellular network between the cementum and alveolar bone. The PDL cellular network is presumed to synchronize PDL fiber bundles and regulate arrays of PDL fiber bundles via gap junctions. In this review, we summarize and discuss our current 3D-histomorphometric studies of the PDL at the mesoscale level. [ABSTRACT FROM AUTHOR]

Herein, the successful use of a focused ion beam/scanning electron microscope to prepare microsamples of radioactive single crystals for X‐ray diffraction analysis is reported. This technique was used to extract and analyze crystalline Pu‐containing particles as small as 28 µm3 from Hanford soil taken from the 216‐Z‐9 waste crib, which were then crystallographically characterized using single‐crystal X‐ray diffraction to confirm the cubic structure of PuO2. As a systematic proof of concept, the technique was first tested using UO2 crystals milled into cubic shapes with approximate volumes of 4620, 1331, 125, 8 and 1 µm3, in order to empirically determine the crystal size limits for characterization by a laboratory‐based diffractometer with a sealed tube Mo or Ag anode X‐ray source and a charge‐coupled device detector. [ABSTRACT FROM AUTHOR]

Some Pyrochroidae species are known as "canthariphilous" for their attraction to cantharidin (CTD), a toxic terpene with anti-predatory effects, produced in nature by only two beetle families (Meloidae and Oedemeridae). It has been demonstrated that males of Neopyrochroa flabellata ingesting CTD are positively selected by females. Indeed, the compound is re-emitted from a glandular cranial apparatus as secretions that are licked up by females during courtship behaviour, inducing copulation. Herein, we provide the first description of the glands associated to the cranial apparatus of male Pyrochroinae using the European species Pyrochroa coccinea as a model. Morphological analyses show that the cranial apparatus consists of a concave pit lined with short setae retaining secretions emitted through numerous glandular pores. Ultrastructural investigations reveal the presence of two different class 3 glands (Gl.A and Gl.B), intermixed at the level of the pit but exhibiting distinct features. Gl.A are mainly characterised by short conducting canals, rounded nuclei and electrondense vesicles while Gl.B are characterised by long conducting canals, irregular nuclei, vesicles containing a particulate substance and a multifolded plasma membrane. Observations of sexual behaviour are also reported for P. coccinea and compared to N. flabellata, confirming the involvement of cranial apparatus secretions in courtship behaviour. • Cranial apparatus glands are analysed for the first time in Pyrochroidae • Two different types of class 3 glands discharge their secretions in the cranial pit. • The two ectodermal glands exhibit specific ultrastructural features and secretions. • We assess which ultrastructural features might be related to cantharidin management. • P. coccinea cranial apparatus is involved in both pre- and post-copulatory behaviour. [ABSTRACT FROM AUTHOR]

• Identification of the self-catalytic characteristics in the pitting cavity. • Discovery of high chloride and no oxygen in the pitting cavity. • Exploration of core-shell chemical and structural distribution in pitting corrosion. • Revelation of the porous core being rich in chloride and the shell being rich in oxygen in pitting corrosion. • Insight into the pitting microstructures with a ferric chloride core and an iron oxide shell. Carbon steel experiences serious pitting corrosion in the atmospheres containing chloride ions. The present research is focused on revealing a three-dimensional chemical and microstructural insight into pitting corrosion with high spatial resolution nano-tomography by addressing 1018 carbon steel after exposed in the marine atmosphere. A well-defined core-shell structure for pitting corrosion products was revealed by using a combination of dual-beam focused ion beam system with scanning electron microscope (FIB/SEM) and high-resolution transmission electron microscope (HRTEM). The core is made of ferric chloride and the shell is constituted with iron oxides. Pitting corrosion is a self-catalytic process and the corrosion products grow to be porous flower-type structures. The porosity in the core provides a diffusion path for the ions. Inclusions promote pitting corrosion growth. [ABSTRACT FROM AUTHOR]

Pathogens survive and propagate within host cells through a wide array of complex interactions. Tracking the molecular and cellular events by multidimensional fluorescence microscopy has been a widespread tool for research on intracellular pathogens. Through major advancements in 3D electron microscopy, intracellular pathogens can also be visualized in their cellular environment to an unprecedented level of detail within large volumes. Recently, multidimensional fluorescence microscopy has been correlated with volume electron microscopy, combining molecular and functional information with the overall ultrastructure of infection events. In this review, we provide a short introduction to correlative focused ion beam/scanning electron microscopy (c-FIB/SEM) tomography and illustrate its utility for intracellular pathogen research through a series of studies on Shigella , Salmonella , and Brucella cellular invasion. We conclude by discussing current limitations of and prospects for this approach. Highlights A current challenge in studying pathogen–host interactions is understanding how molecular players and cellular mechanisms function in the three-dimensional cellular environment at subdiffraction resolution. FIB/SEM is an emerging technique that can be used to investigate expansive three-dimensional cellular interfaces between pathogen and host at ultrastructural resolution. The correlation of FIB/SEM with fluorescence microscopy (c-FIB/SEM) enables the study of transient or rare infection events while also providing molecular specificity within the ultrastructural landscape. Recently, c-FIB/SEM has revealed fundamental aspects of the mechanisms of cellular invasion by Shigella , Salmonella , and Brucella. c-FIB/SEM is becoming an indispensable tool for pathogen research as it can provide a wealth of biological information not obtainable by other technologies. [ABSTRACT FROM AUTHOR]

Bombardier beetles are famous for their unique ability to explosively discharge hot quinones from their pygidial glands when threatened. Here we provide the first detailed description of the ultrastructure of the defensive gland system of the genus Paussus , the most speciose genus in the ground beetle subfamily Paussinae. Paussine beetles are commonly known as "flanged bombardier beetles" due to the presence of a flange on their elytra that assists in directing their defensive chemicals toward the front of their bodies. In this paper, we use optical, fluorescence and focused ion beam (FIB/SEM) microscopy to analyse and illustrate anatomy and ultrastructure of the explosive defensive system of Paussus favieri , a charismatic myrmecophilous species. The defensive system of this species consists of two independent, symmetrical glands each composed of secretory lobes, a long collecting duct, a bilobed reservoir chamber, a cuticular valve, a sclerotized reaction chamber, and an accessory chamber, associated with the reaction chamber, that is surrounded by several isolated glandular cells. Differences between the pygidial defensive systems of Paussus favieri and those of Brachininae are discussed. Image 1 • We analyse the anatomy and ultrastructure of Paussus favieri explosive defensive system. • We use histology, fluorescence and focused ion beam/scanning electron microscopy. • We compare the system of P. favieri with that of the Brachininae bombardier beetles. • We discuss whether the explosive system is a preadaptation for evolution of myrmecophily. [ABSTRACT FROM AUTHOR]

Highlights • Anatomy and ultrastructure of Metriini explosive defensive system is described. • We use dissections, histology and focused ion beam/scanning electron microscopy. • Differences between Metriini and Brachininae explosive systems are highlighted. • Our findings support double evolution of explosive defensive strategy in beetles. • We discuss if explosive system is a preadaptation for evolution of myrmecophagy. Abstract Bombardier beetles, belonging to the carabid subfamilies Paussinae and Brachininae, are famous for their unique ability to explosively discharge a hot spray of quinones from their pygidial glands when threatened. The paussine tribe Metriini is broadly acknowledged as the most basal group of bombardiers. In order to complement the available information on the chemical substances and the primitive discharging mechanism of Metriini, we provide a detailed morpho-functional analysis of the explosive defensive system of Metrius contractus Eschscholtz, 1829 and Sinometrius turnai Wrase and J. Schmidt, 2006, representatives of the two genera in this tribe. We use dissections, histology, scanning electron microscopy (SEM), and focused ion beam microscopy (FIB/SEM) to describe and illustrate various levels of anatomical complexity. FIB/SEM microscopy is used to analyse ultrastructural features of the cellular regions, replacing the classical transmission electron microscopy (TEM). Compared to other Paussinae tribes, Metriini lacks the typical flange of Coanda, the elytral fold used to direct the defensive secretions forward, but has a similar arrangement of internal components. We find that the internal components of the explosive defensive system, including the secretory lobes, collecting duct, reservoir chamber, valve, reaction chamber, accessory chamber and accessory glands, are only slightly different between Metrius Eschscholtz, 1829 and Sinometrius Wrase and J. Schmidt, 2006. The accessory chamber to the reaction chamber is a unique, derived character state common to all Paussinae examined and therefore represents a clear apomorphy of the Paussinae. We use the same microscopy techniques as used in a recent publication on the Brachininae, to compare the defensive systems of Metriini and Brachininae. We find a lack of morphological similarity at the ultrastructural level, suggesting that the bombarding mechanism may have evolved independently in the Paussinae and the Brachininae, perhaps in response to different ecological pressures. [ABSTRACT FROM AUTHOR]

Sliding friction of copper foil surface, which was exposed to bis (6‐acetyl‐1,3,6,2 ‐dioxazaborocan‐2‐yl) methyl phosphate (NEPB) as extreme‐pressure/antiwear agent in base oil while rubbing by a ferroalloy disk under high load, was explored in a multi‐specimen test machine to investigate the wear mechanism of NEPB on thin film lubrication. The results reveal that NEPB reduces the friction coefficient to ~0.16 at normal load of 200 N, and it has better antiwear and friction‐reducing property than the common copper oil. Ferroalloy against copper foil is along the Cu (111) direction, and the friction coefficient decrease relies on the spontaneous formation of chemically nanolayered tribofilm (approx. 30 nm) during sliding wear. The size of wear debris arranges from 6 nm to submicron. Decomposed borate esters and organic nitrides were adsorbed on the worn surface and P element of NEPB as extreme‐pressure/antiwear agent reacted with Cu and existed in the form of phosphates, both of which contributed to the formation of boundary lubricating film. [ABSTRACT FROM AUTHOR]

The marine, bloom-forming dinoflagellate Prorocentrum cordatum CCMP 1329 (formerly P. minimum ) has a genome atypical of eukaryotes, with a large size of ~4.15 Gbp, organized in plentiful, highly condensed chromosomes and packed in a dinoflagellate-specific nucleus (dinokaryon). Here, we apply microscopic and proteogenomic approaches to obtain new insights into this enigmatic nucleus of axenic P. cordatum . High-resolution focused ion beam/scanning electron microscopy analysis of the flattened nucleus revealed highest density of nuclear pores in the vicinity of the nucleolus, a total of 62 tightly packed chromosomes (~0.4-6.7 µm 3 ), and interaction of several chromosomes with the nucleolus and other nuclear structures. A specific procedure for enriching intact nuclei was developed to enable proteomic analyses of soluble and membrane protein-enriched fractions. These were analyzed with geLC and shotgun approaches employing ion-trap and timsTOF (trapped-ion-mobility-spectrometry time-of-flight) mass spectrometers, respectively. This allowed identification of 4,052 proteins (39% of unknown function), out of which 418 were predicted to serve specific nuclear functions; additional 531 proteins of unknown function could be allocated to the nucleus. Compaction of DNA despite very low histone abundance could be accomplished by highly abundant major basic nuclear proteins (HCc2-like). Several nuclear processes including DNA replication/repair and RNA processing/splicing can be fairly well explained on the proteogenomic level. By contrast, transcription and composition of the nuclear pore complex remain largely elusive. One may speculate that the large group of potential nuclear proteins with currently unknown functions may serve yet to be explored functions in nuclear processes differing from those of typical eukaryotic cells. IMPORTANCE Dinoflagellates form a highly diverse group of unicellular microalgae. They provide keystone species for the marine ecosystem and stand out among others by their very large, unusually organized genomes embedded in the nuclei markedly different from other eukaryotic cells. Functional insights into nuclear and other cell biological structures and processes of dinoflagellates have long been hampered by the paucity of available genomic sequences. The here studied cosmopolitan P. cordatum belongs to the harmful algal bloom-forming, marine dinoflagellates and has a recently de novo assembled genome. We present a detailed 3D reconstruction of the P. cordatum nucleus together with comprehensive proteogenomic insights into the protein equipment mastering the broad spectrum of nuclear processes. This study significantly advances our understanding of mechanisms and evolution of the conspicuous dinoflagellate cell biology., Competing Interests: The authors declare no conflict of interest.

Stridulatory organs in the myrmecophilous carabid beetle tribe Paussini have long been recognized and used as a defining character of some genera and higher level taxa, however their morphology has only roughly been described. Here, we describe the fine morphology of Paussini stridulatory organs using scanning electron (SEM) and focused ion beam (FIB) microscopy. Within this tribe, there are three types of stridulatory organs, each with different positions of the scraper (plectrum) and file (pars stridens). Type I (abdomen-femur type) is located on the abdomen (scraper) and metafemur (file) in the subtribe Paussina (sensu Geiselhardt et al., 2007, Naturwissenschaften 94: 871-894). Type II (thorax-femur type) and Type III (femur-thorax type) are located on the mesothorax and mesofemur in two different genera of the subtribe Platyrhopalina, however in Euplatyrhopalus the scraper is located on the mesothorax and the file is located on the mesofemur (Type II), whereas in the genus Platyrhopalopsis the structures are reversed in that the file is located on the mesothorax and the scaper is located on the mesofemur (Type III). The independent evolution of three types of stridulatory organs in three lineages of Paussini suggests that acoustical communication has played an important role in the evolution of ant nest beetles. While the roles of stridulation in this group remain speculative, we verified that all three types of stridulatory organs are present in both sexes and are similar to stridulatory organs known in their host ants which also use stridulation as a method of communication. We discuss the possibility that the beetles stridulation could (1) facilitate their exploitation of ant colonies, and (2) be involved in mate recognition and courtship.

A portfolio is presented documenting economic, high-resolution correlative focused ion beam scanning electron microscopy (FIB/SEM) in routine, comprising: (i) the use of custom-labeled slides and coverslips, (ii) embedding of cells in thin, or ultra-thin resin layers for correlative light and electron microscopy (CLEM) and (iii) the claim to reach the highest resolution possible with FIB/SEM in xyz. Regions of interest (ROIs) defined in light microscope (LM), can be relocated quickly and precisely in SEM. As proof of principle, HeLa cells were investigated in 3D context at all stages of the cell cycle, documenting ultrastructural changes during mitosis: nuclear envelope breakdown and reassembly, Golgi degradation and reconstitution and the formation of the midzone and midbody. [ABSTRACT FROM AUTHOR]

Knowing the proportions of asymmetric (excitatory) and symmetric (inhibitory) synapses in the neuropil is critical for understanding the design of cortical circuits. We used focused ion beam milling and scanning electron microscopy (FIB/SEM) to obtain stacks of serial sections from the six layers of the juvenile rat (postnatal day 14) somatosensory cortex (hindlimb representation). We segmented in three-dimensions 6184 synaptic junctions and determined whether they were established on dendritic spines or dendritic shafts. Of all these synapses, 87-94% were asymmetric and 6-13% were symmetric. Asymmetric synapses were preferentially located on dendritic spines in all layers (80-91%) while symmetric synapses were mainly located on dendritic shafts (62-86%). Furthermore, we found that less than 6% of the dendritic spines establish more than one synapse. The vast majority of axospinous synapses were established on the spine head. Synapses on the spine neck were scarce, although they were more common when the dendritic spine established multiple synapses. This study provides a new large quantitative dataset that may contribute not only to the knowledge of the ultrastructure of the cortex, but also towards defining the connectivity patterns through all cortical layers. [ABSTRACT FROM AUTHOR]

Tin and tin-alloyed electroplated films are known to be susceptible to whisker growth under a range of conditions, many of which result in the generation of compressive stresses in the film. Compressive stress is considered to be one of the primary causes for whisker nucleation and growth. While extensive investigations have been performed on whisker growth, there have been few studies on the mechanical properties of tin whiskers themselves. We report on the tensile behavior of tin whiskers that were obtained by indentation and furnace aging of electroplated tin films on copper disks. Tensile tests of the whiskers were conducted in situ in a dual beam focused ion beam (FIB)-scanning electron microscope (SEM) system using a micro electro-mechanical systems (MEMS) based tensile testing stage. The strength of the whiskers was found to decrease with an increase in gage length and aged whiskers were found to be weaker than their indented counterparts. The observed gage length effect can be attributed to the probability of finding more defects as the whisker length increases. The effect of processing on the observed strength variation was investigated by analyzing the oxygen content in the whiskers via energy dispersive spectroscopy and the microstructure through transmission electron microscopy (TEM). The deformation mechanisms of whiskers were also inferred using post-mortem TEM. It was observed that the whiskers grown by indentation were dislocation free both before and after deformation. In contrast, whiskers grown by aging showed notable dislocation content (arranged in low energy configurations) even before deformation. [ABSTRACT FROM AUTHOR]

Based on serial sectioning, focused ion beam scanning electron microscopy (FIB/SEM), and electron tomography, we depict in detail the highly unusual anatomy of the marine hyperthermophilic crenarchaeon, Ignicoccus hospitalis. Our data support a complex and dynamic endomembrane system consisting of cytoplasmic protrusions, and with secretory function. Moreover, we reveal that the cytoplasm of the putative archaeal ectoparasite Nanoarchaeum equitans can get in direct contact with this endomembrane system, complementing and explaining recent proteomic, transcriptomic and metabolomic data on this inter-archaeal relationship. In addition, we identified a matrix of filamentous structures and/or tethers in the voluminous inter-membrane compartment (IMC) of I. hospitalis, which might be responsible for membrane dynamics. Overall, this unusual cellular compartmentalization, ultrastructure and dynamics in an archaeon that belongs to the recently proposed TACK superphylum prompts speculation that the eukaryotic endomembrane system might originate from Archaea.

This study aims to quantify the effect of heating and freezing temperatures on the pore size distribution of saturated clays. Three kaolinite clay specimens were subjected to different temperatures: 20, 70, and − 10 °C. Upon achieving the desired temperature for each specimen, the specimens were flash frozen in liquid nitrogen to preserve their microstructure. Each specimen was, then, freeze-dried for 24 h after which consecutive two-dimensional (2-D) SEM images were taken using a dual focused ion beam/scanning electron microscope. The produced 2-D images of each specimen were used to reconstruct three-dimensional tomographies of the specimens, which were analyzed to determine the pore size distribution at each temperature. Compared to the specimen at room temperature, the pores in the specimen subjected to − 10 °C were larger; this is believed to be due to the formation of ice lenses inside the pores upon freezing and potential merging between initial pores to form larger pores. On the other hand, the heated specimen showed an increase in the volume of the smaller pores and a decrease in the volume of the larger pores compared to the specimen at room temperature. This opposite behavior between the small and large pores in the heated specimen is justified considering (1) the easier flow of water out of the larger pores compared to that in the smaller pores and (2) the anisotropic nature of the thermal expansion of the clay particles. [ABSTRACT FROM AUTHOR]

Mitochondria undergo morphological changes through fusion and fission for their quality control, which are vital for neuronal function. In this study, we examined three-dimensional morphologies of mitochondria in motor neurons under normal, nerve injured, and nerve injured plus fission-impaired conditions using the focused ion beam/scanning electron microscopy (FIB/SEM), because the FIB/SEM technology is a powerful tool to demonstrate both 3D images of whole organelle and the intra-organellar structure simultaneously. Crossing of dynamin-related protein 1 (Drp1) gene-floxed mice with neuronal injury-specific Cre driver mice, Atf3:BAC Tg mice, allowed for Drp1 ablation specifically in injured neurons. FIB/SEM analysis demonstrated that somatic mitochondrial morphologies in motor neurons were not altered before or after nerve injury. However, the fission impairment resulted in prominent somatic mitochondrial enlargement, which initially induced complex morphologies with round regions and long tubular processes, subsequently causing a decrease in the number of processes and further enlargement of the round regions, which eventually resulted in big spheroidal mitochondria without processes. The abnormal mitochondria exhibited several degradative morphologies: local or total cristae collapse, vacuolization, and mitophagy. These suggest that mitochondrial fission is crucial for maintaining mitochondrial integrity in injured motor neurons, and multiple forms of mitochondria degradation may accelerate neuronal degradation. [ABSTRACT FROM AUTHOR]

Based on serial sectioning, focused ion beam scanning electron microscopy (FIB/SEM), and electron tomography, we depict in detail the highly unusual anatomy of the marine hyperthermophilic crenarchaeon, Ignicoccus hospitalis. Our data support a complex and dynamic endomembrane system consisting of cytoplasmic protrusions, and with secretory function. Moreover, we reveal that the cytoplasm of the putative archaeal ectoparasite Nanoarchaeum equitans can get in direct contact with this endomembrane system, complementing and explaining recent proteomic, transcriptomic and metabolomic data on this inter-archaeal relationship. In addition, we identified a matrix of filamentous structures and/or tethers in the voluminous inter-membrane compartment (IMC) of I. hospitalis, which might be responsible for membrane dynamics. Overall, this unusual cellular compartmentalization, ultrastructure and dynamics in an archaeon that belongs to the recently proposed TACK superphylum prompts speculation that the eukaryotic endomembrane system might originate from Archaea. [ABSTRACT FROM AUTHOR]

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